Functional and physical molecular size of the chicken hepatic lectin determined by radiation inactivation and sedimentation equilibrium analysis.
Radiation inactivation and sedimentation equilibrium analysis were used to determine the functional and physical size of the chicken hepatic membrane receptor that binds N-acetylglucosamine-terminated glycoproteins. Purified plasma membranes from chicken liver were irradiated with high energy electrons and assayed for 125I-agalactoorosomucoid binding. Increasing the dose of ionizing radiation resulted in a monoexponential decay in binding activity due to a progressive loss of binding sites. The molecular mass of the chicken lectin, determined in situ by target analysis, was 69,000 +/- 9,000 Da. When the same irradiated membranes were solubilized in Brij 58 and assayed, the binding protein exhibited a target size of 62,000 +/- 4,000 Da; in Triton X-100, the functional size of the receptor was 85,000 +/- 10,000 Da. Sedimentation equilibrium measurements of the purified binding protein yielded a lower limit molecular weight of 79,000 +/- 7,000. However, the solubilized lectin was detected as a heterogeneous population of oligomers with molecular weights as high as 450,000. Addition of calcium or calcium plus N-acetylglucosamine decreased the higher molecular weight species, but the lower limit molecular weights remained invariant. Similar results were determined when the chicken lectin was solubilized in Brij 58, C12E9, or 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonic acid (CHAPS). Results from the present study suggest that in the plasma membrane, the functional species of the chicken hepatic lectin exists as a trimer. However, in detergent solution, the purified receptor forms a heterogeneous population of irreversible oligomers that exhibit binding activity proportional to size.
Steer
C J
CJ
Laboratory of Biochemistry and Metabolism, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Osborne
J C
JC
Kempner
E S
ES
D000818:Animals; D037263:Asialoglycoprotein Receptor; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002645:Chickens; D008099:Liver / Q000378:metabolism / Q000528:radiation effects; D046911:Macromolecular Substances; D008970:Molecular Weight; D011971:Receptors, Immunologic / Q000302:isolation & purification / Q000378:metabolism* / Q000528:radiation effects
D037263:Asialoglycoprotein Receptor; D046911:Macromolecular Substances; D011971:Receptors, Immunologic